Video Atomic Force Microscopy Shows Real-Time CRISPR Gene Editing
Since 2012, researchers have used a powerful "genome editing" technique called CRISPR to trim, cut, replace, or add biological DNA sequences. CRISPR is derived from the microbial immune system, an engineering editing system that uses an enzyme that cuts a small RNA as a guiding tool into DNA, where it can be cut or otherwise altered.
CRISPR has become the most popular gene editing technology in the life sciences, and its effect has been unanimously recognized. Although scientists can indirectly demonstrate the function of CRISPR by RT-PCR, WB, etc., there is no direct evidence to confirm. The reasons are as follows: First, the interaction rate between biomolecules is fast, and high-speed imaging means is needed to capture; secondly, biomolecules are relatively small, usually nanometer-scale, and ordinary microscopes cannot be distinguished due to optical diffraction limits. Recently, scientists at Kanazawa University in Japan successfully observed real-time CRISPR gene editing using video atomic force microscope HS-AFM, which provided direct evidence for the effectiveness of CRISPR technology.
HS-AFM video results visually show conformational differences:
HS-AFM video results show that apo-Cas9 is a flexible conformations, while Cas9-RNA is a stable bilobed architecture.
Cas9-RNA-mediated PAM-dependent DNA recognition
Cas9-RNA targets the target DNA to form a Cas9-RNA-DNA complex.
Cas9-RNA cuts the DNA of interest
In the presence of Mg2+, Cas9-RNA specifically cleaves the DNA of interest.
The completion of this work mainly relies on the ultra-high-speed video atomic force microscope HS-AFM developed by RIBM Japan. The HS-AFM breaks through the limitation of the traditional atomic force microscope "scanning imaging speed", enabling ultra-fast dynamic imaging in liquid environments. The resolution is at the nano level. The sample to be tested does not need special fixation, does not affect the activity of biomolecules, and is especially suitable for dynamic observation of biological macromolecule interaction. Since its launch, there have been more than 80 users worldwide, and more than 200 SCI papers have been published, including top magazines such as Science, Nature, and Cell.
New product recommendation - HS-AFM came to China
In order to better serve domestic customers, Quantum Design China has introduced this ultra-high-speed video-grade AFM to China. If you have scientific research needs, please contact us!
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